The present invention relates to nucleic acid constructs encoding human GLP-1 peptide and human cells engineered for expression of these nucleic acid constructs to produce GLP-1 peptide and insulin for the treatment and prevention of diabetes, hyperglycemia and weight reduction. The nucleic acid constructs comprise a natural or modified nucleotide sequence which encodes natural human glucagon-like peptide-1 (GLP-1). The modified construct was made using optimized codon usage (Codon Usage Database is available at www.kazusa.or.jp/codon/) and nucleic acid fragment was synthesized commercially (Aptagen, Inc). Preferably, the constructs comprise natural or modified chimeric nucleic acid sequence encoding a human pro-insulin leader, a glucagon-like peptide-1 (GLP-1), and a furin cleavable site between the human pro-insulin leader sequence and the GLP-1. Engineered human cell lines comprising these constructs and which produce natural GLP-1 constitutively by utilizing endogenously produced furin are provided. The engineered cells which produce GLP-1 constitutively may also have a nucleic acid construct encoding furin, which is expressed in a glucose-regulated manner. Also provided are engineered human cells expressing both GLP-1 and proinsulin constitutively and expressing furin in a glucose-dependent manner, wherein the proinsulin and GLP-1 nucleic acid sequences each also encode a furin cleavable site, such that upon furin expression in a glucose-regulated manner, i.e., in the presence of an elevated blood glucose level, cleavage of the proinsulin and GLP-1 chimeric sequences by furin at the respective furin cleavage sites produces insulin and GLP-1. Both types of engineered cells, i.e., the constitutively producing GLP-1 cells; and cells co-producing GLP-1 and insulin constitutively and furin in a glucose-dependent manner, may be used for the treatment and prevention of diabetes, hyperglycemia and weight reduction.
Diabetes mellitus is a group of diseases characterized by high levels of blood glucose. Type I diabetes, previously called insulin-dependent diabetes mellitus (IDDM) or juvenile-onset diabetes, occurs when the immune system destroys pancreatic beta cells, the cells which make the hormone insulin that regulates blood sugar. Type II diabetes, previously called non-insulin dependent diabetes mellitus (NIDDM) or adult-onset diabetes, begins as insulin resistance, i.e., increased hepatic glucose production and decreased insulin-mediated glucose transport at the muscle and adipose tissue level; as the need for insulin rises, the pancreas gradually loses its ability to produce insulin. About 90% to 95% of all diagnosed cases of diabetes are Type II diabetes. This type of diabetes is associated with older age, obesity, family history of diabetes, history of gestational diabetes, impaired glucose metabolism, physical inactivity, and race/ethnicity. Gestational diabetes is characterized by glucose intolerance during pregnancy and occurs more frequently among obese women and those with a family history of diabetes; after pregnancy some women who have had gestational diabetes may develop Type II diabetes. Other types of diabetes occur due to genetic conditions or other factors such as drugs, surgery, malnutrition, infections and other diseases.
GLP-1, an insulinotropic hormone, is secreted postprandially by intestinal L cells as a proteolytic cleavage product of pre-pro-glucagon. It is know as an incretin or gut hormone. GLP-1 has pleiotropic biological effects and the clinical implications of which are very important for type II diabetic patients. GLP-1 has been shown to be a transcriptional inducer of islet cell-specific genes. It stimulates insulin secretion by beta cells in response to an increase in glucose levels and is also responsible for inhibition of glucagon secretion and a decrease in the rate of gastric emptying and acid secretion. GLP-1 has been shown to increase islet cell mass by promoting beta cell neogenesis from ductal cells. The role of GLP-1 in glucose tolerance and the possible involvement of this peptide hormone in the pathogenesis of diabetes makes it a candidate as a new therapeutic agent for people with Type II diabetes. In diabetic patients, a significant induction of insulin secretion and correction of post-prandial hyperglycemia has been achieved by injecting pharmacological, i.e., therapeutically effective, levels of GLP-1 (50-100 pM). Intravenous infusion of GLP-1 in fasted Type II diabetic patients, who were markedly hyperglycemic, completely corrected their blood glucose levels. Thus, GLP-1 appears to be a good therapeutic agent to control hyperglycemia in patients who have Type II diabetes. However, its potential as a new therapeutic agent is limited because this peptide cannot be administered orally, and it has short half life (about 5 minutes or less) in vivo.
GLP-1 is a product of posttranslational processing of the glucagon precursor proglucagon in intestinal L cells and the brain. Other peptide hormones derived from proglucagon include glucagon (in the pancreas) and oxyntomodulin and GLP-2 (in the intestines and brain). There are two forms of full length N-terminal GLP-1, GLP-1 (1-37) and GLP-1 (1-36)amide. Both forms are active and are produced when the GLP-1 polypeptide is cleaved to remove the first six amino acids resulting in the active peptides GLP-1 (7-37), having 31 amino acids, and GLP-1 (7-36) amide, having 30 amino acids. The majority of circulating biologically active GLP-1 is found in the amidated form, GLP-1 (7-36) amide, with lesser amounts of the bioactive non-amidated GLP-1 (7-37) also detectable. The active GLP-1 undergoes rapid degradation by N-terminal cleavage of the first two amino acids (His1-Ala2) by circulating di-peptidyl peptidase IV (DPPIV) resulting in the short half life of GLP-1.
On Apr. 29, 2005, the U.S. Food and Drug Administration approved the first incretin mimetic, BYETTA® (exenatide) injection, as an adjunct therapy for Type II diabetes patients who have not achieved adequate control of blood sugar with two common oral diabetes medications, metformin and/or sulfonylurea. BYETTA® is also indicated as a monotherapy for patients with Type II diabetes. BYETTA® exhibits many of the same effects as the human incretin hormone GLP-1, in regulating blood sugar, according to the manufacturer. Exenatide is a synthetic version of exendin-4, a naturally-occurring hormone, which is a 39-amino acid peptide amide having the amino acid sequence H-His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu -Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2. Exenatide's amino acid sequence partially overlaps that of the human GLP-1, but has a longer half-life than native GLP-1. Exenatide has been shown to bind and activate the known human GLP-1 receptor in vitro.
Analogs of GLP-1, such as extendin-4 or the mutated GLP-1 containing glycine (Gly) as the second amino acid residue, show potent insulinotropic effects and have a longer half life than GLP-1. However, because these molecules are foreign, they cannot be degraded rapidly in the body as native GLP-1. Consequently, a need exists in the field of hyperglycemia treatment, especially in patients with Type II diabetes as well as those having an overweight condition, for nucleic acid constructs that constitutively express human GLP-1, and engineered cells comprising said constructs, to achieve normoglycemia and wherein the produced GLP-1 is degraded when not needed for stimulating insulin production. Nucleic acid constructs which constitutively express nucleic acid sequences comprising human GLP-1 constitutively express nucleic acid constructs comprising proinsulin; and express furin in a glucose-dependent manner, such that an increased level of glucose stimulates furin production, resulting in cleavage at furin cleavable sites of the respective nucleic acid constructs produces GLP-1 and insulin, would provide an alternative form of hyperglycemia treatment, diabetes and overweight conditions. Engineered cells comprising nucleic acid constructs which co-express human GLP-1 and insulin also would provide another therapeutic route for the aforementioned illnesses and/or metabolic conditions.